Phage integrases are enzymes that mediate unidirectional site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB. Integrases may be grouped into two major families, the tyrosine recombinases and the serine recombinases, based on their mode of catalysis.
Tyrosine family integrases, such as lambda integrase, utilize a catalytic tyrosine to mediate strand cleavage, tend to recognize longer attP sequences, and require other proteins encoded by the phage or the host bacteria.
Phage integrases from the serine family are larger, use a catalytic serine for strand cleavage, recognize shorter attP sequences, and do not require host cofactors. Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale.
These properties give phage integrases growing importance for the genetic manipulation of living eukaryotic cells, especially those with large genomes such as mammals and most plants, for which there are few tools for precise manipulation of the genome.
The use of lambda integrases has been subject to extensive research for catalyzing site-specific DNA recombination. For example, two mutant lambda integrases, Int-h (E174K) and its derivative Int-h/218 (E174K/E218K) have been described and were shown to catalyze intermolecular recombination reactions at least as efficiently as the corresponding intramolecular recombination reactions in human cells. Although the presence of arm-site sequences have been shown to increase the recombination of core-sites by Int-h/218 in vivo, given the absence of an attB site in the human genome, recombination reactions occur in non-cognate sites in an essentially random manner.
This makes it difficult to engineer cell lines in a controlled, reproducible fashion.
Therefore, there remains a need to provide mutant integrases having greater efficiency and specificity in catalyzing site specific recombination reactions.